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1.
Methods Enzymol ; 672: 75-102, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934486

RESUMO

Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA. However, when a helicase and DNA polymerase are functionally and physically coupled, they catalyze fast and highly processive leading strand DNA synthesis. DNA polymerase has a 3'-5' exonuclease activity, which removes nucleotides misincorporated in the nascent DNA. DNA synthesis kinetics, processivity, and accuracy are governed by the interplay of the helicase, DNA polymerase, and exonuclease activities within the replisome. This chapter describes quantitative biochemical and biophysical methods to study the coupling of these three critical activities during DNA replication. The methods include real-time quantitation of kinetics of DNA unwinding-synthesis by a coupled helicase-DNA polymerase complex, a 2-aminopurine fluorescence-based assay to map the precise positions of helicase and DNA polymerase with respect to the replication fork junction, and a radiometric assay to study the coupling of DNA polymerase, exonuclease, and helicase activities during processive leading strand DNA synthesis. These methods are presented here with bacteriophage T7 replication proteins as an example but can be applied to other systems with appropriate modifications.


Assuntos
DNA Polimerase Dirigida por DNA , Exonucleases , DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo
2.
DNA Repair (Amst) ; 117: 103369, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35850061

RESUMO

The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be 'intrinsically' proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by 'extrinsic' proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.


Assuntos
DNA Polimerase III , Replicação do DNA , DNA , DNA Polimerase II/metabolismo , DNA Polimerase III/metabolismo , Exonucleases/metabolismo
3.
Cells ; 11(14)2022 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-35883600

RESUMO

Although DNA degradation might seem an unwanted event, it is essential in many cellular processes that are key to maintaining genomic stability and cell and organism homeostasis. The capacity to cut out nucleotides one at a time from the end of a DNA chain is present in enzymes called exonucleases. Exonuclease activity might come from enzymes with multiple other functions or specialized enzymes only dedicated to this function. Exonucleases are involved in central pathways of cell biology such as DNA replication, repair, and death, as well as tuning the immune response. Of note, malfunctioning of these enzymes is associated with immune disorders and cancer. In this review, we will dissect the impact of DNA degradation on the DNA damage response and its links with inflammation and cancer.


Assuntos
Exonucleases , Neoplasias , Morte Celular , DNA , Reparo do DNA , Exonucleases/genética , Exonucleases/metabolismo , Humanos , Inflamação/genética , Neoplasias/genética
4.
J Ethnopharmacol ; 296: 115524, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-35811028

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects. AIM OF THE STUDY: To find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication. MATERIALS AND METHODS: Bioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay. RESULTS: Recombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 µM hypericin, and was completely suppressed by 50 µM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 µM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC50 against HSV-1 F is 2.59 ± 0.08 µM and EC50 against HSV-1 SM44 is 2.94 ± 0.10 µM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 µM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 µM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 µM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1. CONCLUSION: Hypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.


Assuntos
Herpesvirus Humano 1 , Animais , Antracenos , Antivirais/química , Antivirais/farmacologia , Chlorocebus aethiops , Endonucleases , Exonucleases/metabolismo , Exonucleases/farmacologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Perileno/análogos & derivados , Saccharomycetales , Células Vero , Replicação Viral
5.
Biomolecules ; 12(7)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35883419

RESUMO

Molecular chaperones such as Hsp70 and Hsp90 help fold and activate proteins in important signal transduction pathways that include DNA damage response (DDR). Previous studies have suggested that the levels of the mammalian APE2 exonuclease, a protein critical for DNA repair, may be dependent on chaperone activity. In this study, we demonstrate that the budding yeast Apn2 exonuclease interacts with molecular chaperones Ssa1 and Hsp82 and the co-chaperone Ydj1. Although Apn2 does not display a binding preference for any specific cytosolic Hsp70 or Hsp90 paralog, Ssa1 is unable to support Apn2 stability when present as the sole Ssa in the cell. Demonstrating conservation of this mechanism, the exonuclease APE2 also binds to Hsp70 and Hsp90 in mammalian cells. Inhibition of chaperone function via specific small molecule inhibitors results in a rapid loss of APE2 in a range of cancer cell lines. Taken together, these data identify APE2 and Apn2 as clients of the chaperone system in yeast and mammalian cells and suggest that chaperone inhibition may form the basis of novel anticancer therapies that target APE2-mediated processes.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfatases , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Exodesoxirribonucleases , Exonucleases/metabolismo , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nat Protoc ; 17(5): 1306-1331, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35379945

RESUMO

DNA fluorescence in situ hybridization (FISH) has been a central technique in advancing our understanding of how chromatin is organized within the nucleus. With the increasing resolution offered by super-resolution microscopy, the optimal maintenance of chromatin structure within the nucleus is essential for accuracy in measurements and interpretation of data. However, standard 3D-FISH requires potentially destructive heat denaturation in the presence of chaotropic agents such as formamide to allow access to the DNA strands for labeled FISH probes. To avoid the need to heat-denature, we developed Resolution After Single-strand Exonuclease Resection (RASER)-FISH, which uses exonuclease digestion to generate single-stranded target DNA for efficient probe binding over a 2 d process. Furthermore, RASER-FISH is easily combined with immunostaining of nuclear proteins or the detection of RNAs. Here, we provide detailed procedures for RASER-FISH in mammalian cultured cells to detect single loci, chromatin tracks and topologically associating domains with conventional and super-resolution 3D structured illumination microscopy. Moreover, we provide a validation and characterization of our method, demonstrating excellent preservation of chromatin structure and nuclear integrity, together with improved hybridization efficiency, compared with classic 3D-FISH protocols.


Assuntos
Núcleo Celular , Cromatina , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/genética , DNA/metabolismo , Exonucleases/metabolismo , Hibridização in Situ Fluorescente/métodos , Interfase , Mamíferos
7.
BMC Mol Cell Biol ; 23(1): 17, 2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35399070

RESUMO

BACKGROUND: We have previously found that, in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5'-end were resistant to 5'-monophosphate requiring 5' → 3″ exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them. RESULTS: We now show the production of such 18S and 25S rRNAs in Saccharomyces cerevisiae that have been permanently switched to RNAP II (due to deletion of part of RNAP I upstream activator alone, or in combination with deletion of one component of RNAP I itself). They contain more than one phosphate at their 5'-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to have been modified in the cytoplasm. CONCLUSIONS: Our data confirm the existence of such molecules and firmly establish RNAP II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.


Assuntos
RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exonucleases/metabolismo , Fosfatos/metabolismo , RNA Polimerase II/metabolismo , RNA Ribossômico/genética , RNA Ribossômico 18S , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Vet Res ; 53(1): 32, 2022 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-35461299

RESUMO

The NF-κB pathway is an essential signalling cascade in the defence against viral infections, including African swine fever virus (ASFV) infection. ASFV encodes more than 151 proteins via its own transcription machinery and possesses a great capacity to evade or subvert antiviral innate immune responses. Although some of these viral proteins have been reported, many remain unknown. Here, we show that pD345L, an ASFV-encoded lambda-like exonuclease, acts as an inhibitor of cGAS/STING-mediated NF-κB signalling by blocking the IkappaB kinase (IKKα/ß) activity. Specifically, we showed that overexpression of pD345L suppresses cGAS/STING-induced IFNß and NF-κB activation, resulting in decreased transcription of IFNß and several proinflammatory cytokines, including IL-1α, IL-6, IL-8, and TNFα. In addition, we showed that pD345L acts at or downstream of IKK and upstream of p65. Importantly, we found that pD345L associates with the KD and HLH domains of IKKα and the LZ domain of IKKß and thus interrupts their kinase activity towards the downstream substrate IκBα. Finally, we showed that pD345L-mediated inhibition of NF-κB signalling was independent of its exonuclease activity. Considering these results collectively, we concluded that pD345L blocks IKKα/ß kinase activity via protein-protein interactions and thus disrupts cGAS/STING-mediated NF-κB signalling.


Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Vírus da Febre Suína Africana/fisiologia , Animais , Exonucleases/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Nucleotidiltransferases/metabolismo , Suínos
9.
Talanta ; 243: 123350, 2022 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-35272156

RESUMO

As one of the initiating DNA glycosylases in the base excision repair pathway, Uracil-DNA glycosylase (UDG) plays a pivotal role in maintaining genomic integrity. The abnormal expression of UDG in the organism is highly relevant to multiple diseases. Thus, rapid and sensitive detection of UDG activity is essential to aid early clinical diagnosis and biomedical research. Here we developed a rapid, sensitive and selective biosensor for UDG activity detection based on the substrate preference of Lambda exonuclease (λ exo). The protruding end in the substrate produced by UDG could be digested at a markedly high rate by λ exo, generating a detectable fluorescence signal. This proposed strategy for UDG detection exhibited high selectivity and high sensitivity (0.0001 U/mL) in a short time. It has also been successfully applied to detect UDG in real biological samples and the screening of UDG inhibitors.


Assuntos
Técnicas Biossensoriais , Uracila-DNA Glicosidase , Reparo do DNA , Exonucleases/metabolismo
10.
Life Sci Alliance ; 5(6)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35292537

RESUMO

Mre11 is a versatile exo-/endonuclease involved in multiple aspects of DNA replication and repair, such as DSB end processing and checkpoint activation. We previously demonstrated that forced mitotic entry drives replisome disassembly at stalled replication forks in Xenopus egg extracts. Here, we examined the effects of various chemical inhibitors using this system and discovered a novel role of Mre11 exonuclease activity in promoting mitotic entry under replication stress. Mre11 activity was necessary for the initial progression of mitotic entry in the presence of stalled forks but unnecessary in the absence of stalled forks or after mitotic entry. In the absence of Mre11 activity, mitotic CDK was inactivated by Wee1/Myt1-dependent phosphorylation, causing mitotic exit. An inhibitor of Wee1/Myt1 or a nonphosphorylatable CDK1 mutant was able to partially bypass the requirement of Mre11 for mitotic entry. These results suggest that Mre11 exonuclease activity facilitates the processing of stalled replication forks upon mitotic entry, which attenuates the inhibitory pathways of mitotic CDK activation, leading to irreversible mitotic progression and replisome disassembly.


Assuntos
Replicação do DNA , Exonucleases , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética
11.
Int J Biol Macromol ; 204: 617-626, 2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35150781

RESUMO

RecJ is ubiquitous in bacteria and Archaea, and play an important role in DNA replication and repair. Currently, our understanding on biochemical function of archaeal RecJ is incomplete due to the limited reports. The genome of the hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes one putative RecJ protein (Tga-RecJ). Herein, we report biochemical characteristics and catalytic mechanism of Tga-RecJ. Tga-RecJ can degrade ssDNA in the 5'-3' direction at high temperature as observed in Thermococcus kodakarensis RecJ and Pyrococcus furiosus RecJ, the two closest homologs of the enzyme. In contrasted to P. furiosus RecJ, Tga-RecJ lacks 3'-5' ssRNA exonuclease activity. Furthermore, maximum activity of Tga-RecJ is observed at 50 °C ~ 70 °C and pH 7.0-9.0 with Mn2+, and the enzyme is the most thermostable among the reported RecJ proteins. Additionally, the rates for hydrolyzing ssDNA by Tga-RecJ were estimated by kinetic analyses at 50 °C ~ 70 °C, thus revealing its activation energy (10.5 ± 0.6 kcal/mol), which is the first report on energy barrier for ssDNA degradation by RecJ. Mutational studies showed that the mutations of residues D36, D83, D105, H106, H107 and D166 in Tga-RecJ to alanine almost completely abolish its activity, thereby suggesting that these residues are essential for catalysis.


Assuntos
Proteínas Arqueais , Pyrococcus furiosus , Thermococcus , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Replicação do DNA , DNA de Cadeia Simples/genética , Exonucleases/metabolismo , Pyrococcus furiosus/genética , Thermococcus/genética
12.
Commun Biol ; 5(1): 154, 2022 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-35194144

RESUMO

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Exonucleases/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Anilidas/farmacologia , Animais , Sequência de Bases , Benzimidazóis/farmacologia , COVID-19/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Sinergismo Farmacológico , Exonucleases/genética , Exonucleases/metabolismo , Humanos , Prolina/farmacologia , Pirrolidinas/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Valina/farmacologia , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
13.
RNA ; 28(5): 657-667, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35140172

RESUMO

The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Exonucleases/genética , Exonucleases/metabolismo , Exorribonucleases/metabolismo , Proteínas Nucleares/genética , RNA/genética , RNA/metabolismo , RNA Ribossômico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcriptoma/genética
14.
J Gynecol Oncol ; 33(3): e27, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35128857

RESUMO

OBJECTIVE: The need to perform genetic sequencing to diagnose the polymerase epsilon exonuclease (POLE) subtype of endometrial cancer (EC) hinders the adoption of molecular classification. We investigated clinicopathologic and protein markers that distinguish the POLE from the copy number (CN)-low subtype in EC. METHODS: Ninety-one samples (15 POLE, 76 CN-low) were selected from The Cancer Genome Atlas EC dataset. Clinicopathologic and normalized reverse phase protein array expression data were analyzed for associations with the subtypes. A logistic model including selected markers was constructed by stepwise selection using area under the curve (AUC) from 5-fold cross-validation (CV). The selected markers were validated using immunohistochemistry (IHC) in a separate cohort. RESULTS: Body mass index (BMI) and tumor grade were significantly associated with the POLE subtype. With BMI and tumor grade as covariates, 5 proteins were associated with the EC subtypes. The stepwise selection method identified BMI, cyclin B1, caspase 8, and X-box binding protein 1 (XBP1) as markers distinguishing the POLE from the CN-low subtype. The mean of CV AUC, sensitivity, specificity, and balanced accuracy of the selected model were 0.97, 0.91, 0.87, and 0.89, respectively. IHC validation showed that cyclin B1 expression was significantly higher in the POLE than in the CN-low subtype and receiver operating characteristic curve of cyclin B1 expression in IHC revealed AUC of 0.683. CONCLUSION: BMI and expression of cyclin B1, caspase 8, and XBP1 are candidate markers distinguishing the POLE from the CN-low subtype. Cyclin B1 IHC may replace POLE sequencing in molecular classification of EC.


Assuntos
Neoplasias do Endométrio , Exonucleases , Caspase 8/genética , Caspase 8/metabolismo , Ciclina B1/genética , Ciclina B1/metabolismo , Variações do Número de Cópias de DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Feminino , Humanos , Mutação
15.
Int J Biochem Cell Biol ; 144: 106171, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35093572

RESUMO

B-family DNA polymerases, which are found in eukaryotes, archaea, viruses, and some bacteria, participate in DNA replication and repair. Starting from the N-terminus of archaeal and bacterial B-family DNA polymerases, three domains include the N-terminal, exonuclease, and polymerase domains. The N-terminal domain of the archaeal B-family DNA polymerase has a conserved deoxyuracil-binding pocket for specially binding the deoxyuracil base on DNA. The exonuclease domain is responsible for removing the mismatched base pair. The polymerase domain is the core functional domain and takes a highly conserved structure composed of fingers, palm and thumb subdomains. Previous studies have demonstrated that the thumb subdomain mainly functions as a DNA-binding element and has coordination with the exonuclease domain and palm subdomain. To further elucidate the possible functions of the thumb subdomain of archaeal B-family DNA polymerases, the thumb subdomain of Pyrococcus furiosus DNA polymerase was mutated, and the effects on three activities were characterized. Our results demonstrate that the thumb subdomain participates in the three activities of archaeal B-family DNA polymerases as a common structural element. Both the N-terminal deoxyuracil-binding pocket and thumb subdomain are critical for deoxyuracil binding. Moreover, the thumb subdomain assists DNA polymerization and hydrolysis reactions, but it does not contribute to the fidelity of DNA polymerization.


Assuntos
Pyrococcus furiosus , Sequência de Aminoácidos , DNA/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/química , Exonucleases/metabolismo , Hidrólise , Modelos Moleculares , Nucleotídeos , Polimerização , Estrutura Terciária de Proteína , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Polegar
16.
ACS Synth Biol ; 11(2): 732-746, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35034449

RESUMO

The use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions across two different laboratories. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and cost-effective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.


Assuntos
Escherichia coli , Exonucleases , Sistema Livre de Células/metabolismo , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Exonucleases/metabolismo
17.
Chem Commun (Camb) ; 57(99): 13415-13428, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34796887

RESUMO

Single-molecule fluorescence imaging is among the most advanced analytical technologies and has been widely adopted for biosensing due to its distinct advantages of simplicity, rapidity, high sensitivity, low sample consumption, and visualization capability. Recently, a variety of nucleic acid amplification approaches have been developed to provide a straightforward and highly efficient way for amplifying low abundance target signals. The integration of single-molecule fluorescence imaging with nucleic acid amplification has greatly facilitated the construction of various fluorescent biosensors for in vitro and in vivo detection of DNAs, RNAs, enzymes, and live cells with high sensitivity and good selectivity. Herein, we review the advances in the development of fluorescent biosensors by integrating single-molecule fluorescence imaging with nucleic acid amplification based on enzyme (e.g., DNA polymerase, RNA polymerase, exonuclease, and endonuclease)-assisted and enzyme-free (e.g., catalytic hairpin assembly, entropy-driven DNA amplification, ligation chain reaction, and hybridization chain reaction) strategies, and summarize the principles, features, and in vitro and in vivo applications of the emerging biosensors. Moreover, we discuss the remaining challenges and future directions in this area. This review may inspire the development of new signal-amplified single-molecule biosensors and promote their practical applications in fundamental and clinical research.


Assuntos
Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , Imagem Óptica , DNA/análise , DNA/genética , DNA Polimerase Dirigida por DNA/análise , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Endonucleases/análise , Endonucleases/genética , Endonucleases/metabolismo , Exonucleases/análise , Exonucleases/genética , Exonucleases/metabolismo , Humanos , RNA/análise , RNA/genética , RNA Polimerase Dependente de RNA/análise , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo
18.
Nat Commun ; 12(1): 6811, 2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34815410

RESUMO

Tuning colloidal structure formation is a powerful approach to building functional materials, as a wide range of optical and viscoelastic properties can be accessed by the choice of individual building blocks and their interactions. Precise control is achieved by DNA specificity, depletion forces, or geometric constraints and results in a variety of complex structures. Due to the lack of control and reversibility of the interactions, an autonomous oscillating system on a mesoscale without external driving was not feasible until now. Here, we show that tunable DNA reaction circuits controlling linker strand concentrations can drive the dynamic and fully reversible assembly of DNA-functionalized micron-sized particles. The versatility of this approach is demonstrated by programming colloidal interactions in sequential and spatial order to obtain an oscillatory structure formation process on a mesoscopic scale. The experimental results represent an approach for the development of active materials by using DNA reaction networks to scale up the dynamic control of colloidal self-organization.


Assuntos
Coloides/química , DNA/química , Proteínas de Bactérias/metabolismo , Coloides/metabolismo , DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonuclease I/metabolismo , Exonucleases/metabolismo , Biologia Sintética/métodos
19.
Nat Commun ; 12(1): 5874, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620855

RESUMO

Phospholipase D3 (PLD3) and PLD4 polymorphisms have been associated with several important inflammatory diseases. Here, we show that PLD3 and PLD4 digest ssRNA in addition to ssDNA as reported previously. Moreover, Pld3-/-Pld4-/- mice accumulate small ssRNAs and develop spontaneous fatal hemophagocytic lymphohistiocytosis (HLH) characterized by inflammatory liver damage and overproduction of Interferon (IFN)-γ. Pathology is rescued in Unc93b13d/3dPld3-/-Pld4-/- mice, which lack all endosomal TLR signaling; genetic codeficiency or antibody blockade of TLR9 or TLR7 ameliorates disease less effectively, suggesting that both RNA and DNA sensing by TLRs contributes to inflammation. IFN-γ made a minor contribution to pathology. Elevated type I IFN and some other remaining perturbations in Unc93b13d/3dPld3-/-Pld4-/- mice requires STING (Tmem173). Our results show that PLD3 and PLD4 regulate both endosomal TLR and cytoplasmic/STING nucleic acid sensing pathways and have implications for the treatment of nucleic acid-driven inflammatory disease.


Assuntos
DNA/metabolismo , Exonucleases/genética , Exonucleases/metabolismo , Inflamação/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , RNA/metabolismo , Animais , Células Dendríticas , Endossomos/metabolismo , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Receptores Toll-Like , Transcriptoma
20.
Phys Rev E ; 104(3-1): 034417, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34654207

RESUMO

Being a dual purpose enzyme, the DNA polymerase is responsible for elongation of the newly formed DNA strand as well as cleaving the erroneous growth in case of a misincorporation. The efficiency of replication depends on the coordination of the polymerization and exonuclease activity of DNA polymerase. Here, we propose and analyze a minimal kinetic model of DNA replication and determine exact expressions for the velocity of elongation and the accuracy of replication. We first analyze the case without exonuclease activity. In that case, accuracy is determined by a kinetic competition between stepping and unbinding, with discrimination between correct and incorrect nucleotides in both transitions. We then include exonuclease activity and ask how different modes of additional discrimination in the exonuclease pathway can improve the accuracy while limiting the detrimental effect of exonuclease on the speed of replication. In this way, we ask how the kinetic parameters of the model have to be set to coordinate the two activities of the enzyme for high accuracy and high speed. The analysis also shows that the design of a replication system does not universally have to follow the speed-accuracy trade-off rule, although it does in the biologically realized parameter range. The accuracy of the process is mainly controlled by the crucial role of stepping after erroneous incorporation, which has impact on both polymerase and exonuclease activities of DNA polymerase.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Cinética
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